CN104313177A - Molecular detection method for rapidly identifying carbendazim-resistant genotype-F200Y botrytis cinerea strain - Google Patents
Molecular detection method for rapidly identifying carbendazim-resistant genotype-F200Y botrytis cinerea strain Download PDFInfo
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Abstract
The invention discloses a molecular detection method for rapidly identifying a carbendazim-resistant genotype-F200Y botrytis cinerea strain. The molecular detection method is a molecular detection technology, which is established based on a LAMP (loop-mediated isothermal amplification) technology and is rapid, convenient, high in specificity and sensitivity and low in cost. The detection method comprises the following steps of designing two pairs of specific primers on a beta tubulin of a detected sample, performing LAMP, and judging whether the detected sample is a carbendazim-resistant genotype-F200Y botrytis cinerea strain or not according to the color of a reaction product; if the LAMP product is sky-blue and an electrophoretogram is a ladder band, determining that the product amplification exists and that the detected sample is the carbendazim-resistant genotype-F200Y botrytis cinerea strain; if the LAMP product is purple and the electrophoretogram is free of band, determining that the product amplification does not exist and that the detected sample is a carbendazim-nonresistant genotype-F200Y botrytis cinerea strain. The method is convenient, rapid, low in cost and significant for theoretically guiding the resistance risk evaluation and reasonable medication of botrytis of crops.
Description
Technical field
The present invention is based on loop-mediated isothermal amplification technology (loop-mediated isothermal amplification, LAMP) to the rapid molecular authentication method of the carbendazim resistance gene type F200Y bacterial strain of the pathogen of Botrytis cinerea, the biological detection method of field of biology Middle molecule is belonged to.
Background technology
Gray mold is the fungal diseases of plants that a class is caused by Botrytis cinerea (Botrytis cinerea), the cash crop that 200 various vegetables, fruit tree and ornamental plant etc. are important can be endangered, the generation of this disease can cause plant seedlings, the dampinging off of fruit and storage organ, falls leaves, spend corruption, decayed fruit and rotten kiln, causes serious financial loss.In recent years, the development of producing along with vegetable at protected field, has increased the weight of the generation of gray mold and popular.At present because crop germplasm resource lacks the kind of high botrytis resistant, chemical agent is adopted to be control gray mold one of approach the most easily.For many years, gray mold mainly adopts benzimidazole germicide or prevents and treats based on the built agent of this kind of medicament.But the increase of the using dosage along with the growth of working life, field creates drug-resistant populations gradually, thus causes this sick preventive effect significantly to decline.Through years of researches, Agricultural University Of Nanjing's sterilant biology laboratory finds that the pathogen of Botrytis cinerea is mainly suddenlyd change by the pathogen of Botrytis cinerea 'beta '-tubulin gene (BC1G_00122) to the drug-fast strain of derosal and causes, the sudden change of this genes encoding the 198th or 200 amino acids codons can cause the pathogen of Botrytis cinerea to the generation of carbendazim-resistance, wherein the sudden change of 198 amino acids can cause the pathogen of Botrytis cinerea to show high-level resistance to derosal, and the sudden change of 200 amino acids can cause the pathogen of Botrytis cinerea to derosal performance medium level resistance.200 amino acids mutated-genotype are TTC → TAC, i.e. phenylalanine (Phe, F) → TYR (Tyr, Y), is abbreviated as F200Y.
Traditional authentication method needs separation and Culture pathogenic bacteria, then cultivates on pastille substratum, then differentiates according to the restraining effect of medicament to mycelial growth, the method is long for qualification cycle, reach 1 week, even several weeks from being separated to qualification, and there is living contaminants in Bacteria culturing process.In recent years, along with the genotypic qualification of pathogen resistance, round pcr is widely used, this technology needs expensive test apparatus and loaded down with trivial details electrophoresis process, also need to contact a large amount of poisonous and harmful reagent in qualification process, larger potential safety hazard is existed to experiment operator, and reaches a few hours when identifying.In order to overcome these shortcomings, one receives certain concern at the pathogen of Botrytis cinerea in the qualification of the resistant genotype of derosal based on loop-mediated isothermal amplification technology (LAMP).
Loop-mediated isothermal amplification reaction (LAMP) is the constant temperature nucleic acid Amplification Technologies of a kind of novelty that 2000 are invented by Japanese scholars Notomi etc., is widely used in the gene diagnosis of the disease such as animal, plant.This know-why is: utilize a set of (4 kinds) Auele Specific Primer, self-circulation strand replacement reaction is caused under a kind of effect of high reactivity strand displacement archaeal dna polymerase, in 60 ~ 65 DEG C of scope 60min, while a large amount of synthesis target dna, being attended by by product---the magnesium pyrophosphate precipitation of white produces.Hydroxynaphthol blue (HNB) is a kind of Metal ion indicator, distinct colors is presented according to the change of magnesium ion in reaction solution, being purple time negative (not amplifying product), is sky blue time positive (having product amplification).The maximum feature of LAMP method realizes constant-temperature amplification exactly, does not need the instrument of the costlinesses such as circulating instrument; Amplified reaction is exceedingly fast, and generally completes in 1 hour; The product amount that amplification produces is large, gets final product result of determination, do not need loaded down with trivial details electrophoresis process by naked eyes; Highly sensitive, high specificity; Easy and simple to handle, quick, pole is suitable for the Rapid identification of cause of disease mutated-genotype.
The present invention on the basis that drug resistance mechanism is studied, based on LAMP technology can fast, precise Identification the pathogen of Botrytis cinerea is to the resistant genotype F200Y of derosal.This authentication method has simply, fast, cost is low, and susceptibility high, can improve detection efficiency greatly, has important practical significance to the resistance management of gray mold and the popular early warning of resistance.But, through retrieving the relevant report that at home and abroad there is no the pathogen of Botrytis cinerea at present and identify the LAMP rapid molecular of carbendazim resistance gene type F200Y.
Summary of the invention
The object of the invention is existing the pathogen of Botrytis cinerea there is time-consuming, effort, cost is high, accuracy is low shortcoming in the authentication method of carbendazim resistance bacterial strain, providing a kind of easy, quick, time saving and energy saving, highly sensitive, qualification the pathogen of Botrytis cinerea that testing cost is low to the molecular detecting method of carbendazim resistance gene type F200Y.
Rapid identification the pathogen of Botrytis cinerea of the present invention is to the molecular biology method of carbendazim resistance gene type F200Y bacterial strain, and step is:
(1) on the beta tubulin gene (BC1G_00122) of the pathogen of Botrytis cinerea, comprise the sequences Design LAMP primer of 200 amino acids, respectively with the genomic dna of sensitive strain and resistant genotype F200Y bacterial strain for template, LAMP primer described in utilization is under constant temperature, carry out LAMP amplification, wherein: described LAMP primer respectively:
LAMP reacts outer primer pair used:
F3：ATCGCCAAAGGTTTCCGATA
B3：AGGTGGTAACACCGGACAT
LAMP reacts inner primer used to (containing base mismatch):
FIP：T
GGTCTCGTCAGAGTTCTCAACCAGTTGTCGAGCCATATAACGCA
BIP：TGCATGAGAACCTTGAAGCTCAGCGACGGCGGAAACCAAGTG
(2) its colour-change is observed to above-mentioned LAMP amplified production, and be separated on 3.0% agarose gel electrophoresis, observe amplification;
(3) identify to be whether the Botrytis cinerea bacterial strain of carbendazim resistance gene type F200Y; LAMP amplified production display sky blue, electrophoretogram should be scalariform band, is judged to be the Botrytis cinerea bacterial strain of resistant genotype F200Y; Amplified production is purple, and electrophoretogram increases without band, is judged to be the Botrytis cinerea bacterial strain of non-resistance genotype F200Y.
Wherein:
LAMP cumulative volume described in step (1) is preferably 10 μ L:
LAMP reaction parameter described in step (1) is preferably: 60 DEG C of 45min, 80 DEG C of 10min.
The present invention adopts LAMP technology, and Rapid identification the pathogen of Botrytis cinerea, to the resistant genotype F200Y bacterial strain of derosal, enriches the technical system that the pathogen of Botrytis cinerea detects carbendazim resistance; For the pathogen of Botrytis cinerea resistance monitoring and resistance risk evaluation is provided fundamental basis and technical support, to the happening and prevelence of China's gray mold and resistance management, there is important theory directive significance.
Rapid identification the pathogen of Botrytis cinerea provided by the invention, to the molecular biology method of carbendazim resistance gene type F200Y bacterial strain, has the features such as highly sensitive, high specificity, and compared with prior art, beneficial outcomes of the present invention is:
1, simple and easy to do: this detection method is by thermostat water bath or have the equipment of stable thermal source just can test, and gets final product result of determination by reaction product colour-change, eliminate expensive plant and instrument and loaded down with trivial details electrophoresis process;
2, detection efficiency is high: this detection method substantially increased detection efficiency less than 1 hour detection time used, and traditional indoor bioassay method needs several days time, and PCR detects needs loaded down with trivial details electrophoresis process, also wants a few hours;
3, highly sensitive: the plasmid vector of structure is diluted to different concentration, as template, carry out sensitivity technique, lowest detection lower limit is 100 times of regular-PCR Monitoring lower-cut;
4, high specificity: the method is by 6 isolated areas on 2 pairs of primer specificity identification target sequences, and for 2 isolated areas of PCR primer identification target sequence, specificity improves greatly, the probability that false positive occurs also decreases;
5, accuracy is high: the method is subject to the impact of a large amount of foreign DNA and the impurity existed in reaction mixture hardly, does not need purify DNA from sample, incidence tissue and invalid body directly can be utilized to extract DNA and carry out rapid detection, greatly improve accuracy in detection;
6, the present invention utilizes LAMP technology qualification the pathogen of Botrytis cinerea to carbendazim resistance gene type F200Y bacterial strain both at home and abroad first, this method is simple and efficient, the Accurate Diagnosis of antagonism bacterial strain, understands resistance populations development trend in time, instructs Scientific Usage of Drugs, reduces costs and reduce environmental pollution and have important practical significance.
Accompanying drawing explanation
Fig. 1: the LAMP specific detection detected
Embodiment
Embodiment 1 LAMP reaction system optimization
In order to save appraisal cost, ensure stability and the reliability of this authentication method, to BstDNA polysaccharase in reaction system (8U/ μ L) (0.8-4.0U), Mg
2+(25mM) concentration (0.6-2.0 μ L), primers F IP/BIP (40 μMs) and F3/B3 (10 μMs) concentration (0.1-0.5 μ L), trimethyl-glycine (8M) concentration (0.4-2.0 μ L), HNB (2.5mM) concentration (0.4-1.2 μ L) are optimized, determining optimal reaction system is: BstDNA polysaccharase (8U/ μ L) 0.25 μ L, 10 × ThermoPol1 μ L, MgCl
2(25mM) 1.6 μ L, dNTP (10mM) 0.8 μ L, FIP (40 μMs) 0.4 μ L, BIP (40 μMs) 0.4 μ L, F3 (10 μMs) 0.4 μ L, B3 (10 μMs) 0.4 μ L, trimethyl-glycine (8M) 1.0 μ L, HNB (2.5mM) 0.8 μ L, genomic dna 0.4 μ L, aseptic ddH
2o 2.55 μ L.
Embodiment 2 LAMP reaction parameter is optimized
In order to obtain the suitableeest temperature of reaction and time, ensure the high efficiency of this detection method, the temperature of reaction in reaction parameter and time are optimized, show that optimal reaction temperature and time are respectively 60 DEG C and 45min.
Embodiment 3 LAMP reaction sensitivity is tested
In order to determine the Monitoring lower-cut that LAMP reacts, the DNA fragmentation containing 200 mutational sites of the pathogen of Botrytis cinerea to carbendazim resistance gene type F200Y bacterial strain is cloned into carrier pEASY-T3, transformation of E. coli, picking positive transformant, after extracting plasmid, using 10 times of gradient dilutions as template, carry out LAMP and pcr amplification respectively.Finally draw, the lowest detection lower limit of LAMP is 100 times of regular-PCR Monitoring lower-cut.
Embodiment 4 LAMP atopic is tested
Be that template carries out LAMP amplification respectively with the genomic dna of the pathogen of Botrytis cinerea to different resistant genotype bacterial strain (E198A, E198K, E198V, F200Y) of derosal and wild type strain.When template is genotype F200Y bacterial strain, reaction product color is sky blue, and electrophoretogram becomes scalariform band; When template be wild type strain and other resistant genotype bacterial strain time, reaction product color is purple, and electrophoresis is without band, and the above results shows this authentication method reliable results, high specificity (Fig. 1).
Embodiment 5 LAMP reacts replica test
Be that template carries out LAMP detection to gathering the genomic dna of 8 the pathogen of Botrytis cinereas to carbendazim resistance gene type F200Y bacterial strain of diverse geographic location, take wild-type as contrast, the reaction color that result shows 8 laboratory samples is sky blue, and electrophoretogram is all in scalariform band.And show through the sequencing result of mutator gene, 8 samples are all in 200 origination point sudden changes of beta tubulin sequence.The above results shows this authentication method reliable results, reproducible.
The LAMP method energy that the present invention sets up is accurate, Rapid identification goes out Botrytis cinerea to carbendazim resistance gene type F200Y bacterial strain, for scientific research and production practice provide a kind of authenticate technology easy, quick, with low cost, also be the dynamic monitoring of gray mold to carbendazim resistance colony, the popular early warning of resistance disease and rational use of drug provide theoretical basis and technical director.
Claims (3)
1. based on LAMP technology Rapid identification the pathogen of Botrytis cinerea to a molecular detecting method for carbendazim resistance gene type F200Y bacterial strain, step is:
(1) use two pairs of Auele Specific Primers LAMP constant-temperature amplification is carried out to the genomic dna of testing sample, the base sequence of two pairs of wherein said Auele Specific Primers respectively:
LAMP reacts outer primer pair used:
F3：ATCGCCAAAGGTTTCCGATA
B3：AGGTGGTAACACCGGACAT
LAMP reacts inner primer pair used:
FIP：T
GGTCTCGTCAGAGTTCTCAACCAGTTGTCGAGCCATATAACGCA
BIP：TGCATGAGAACCTTGAAGCTCAGCGACGGCGGAAACCAAGTG
(2) its colour-change is observed to above-mentioned LAMP amplified production, and be separated on 3.0% agarose gel electrophoresis, observe amplification;
(3) identify to be whether the Botrytis cinerea bacterial strain of carbendazim resistance gene type F200Y; LAMP amplified production display sky blue, electrophoretogram should be scalariform band, is judged to be the Botrytis cinerea bacterial strain of resistant genotype F200Y; Amplified production is purple, and electrophoretogram increases without band, is judged to be the Botrytis cinerea bacterial strain of non-resistance genotype F200Y.
2. Rapid identification the pathogen of Botrytis cinerea, to the molecular detecting method of carbendazim resistance gene type F200Y bacterial strain, is characterized in that according to claim 1: the LAMP cumulative volume described in step (1) is preferably 10 μ L:
3. Rapid identification the pathogen of Botrytis cinerea, to the molecular detecting method of carbendazim resistance gene type F200Y bacterial strain, is characterized in that according to claim 1: the LAMP reaction parameter described in step (1) is preferably 60 DEG C of 45min, 80 DEG C of 10min.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104911258A (en) * | 2015-04-23 | 2015-09-16 | 南京农业大学 | Carbendazim resistant genotype F200Y sclerotinia sclerotiorum LAMP detection primer composition and LAMP test kit and LAMP test method |
| CN108315472A (en) * | 2018-04-27 | 2018-07-24 | 安徽省农业科学院植物保护与农产品质量安全研究所 | It is a kind of to be used for the Primer composition and its application that alternaric bacteria LAMP is quickly detected |
| CN109666754A (en) * | 2018-11-26 | 2019-04-23 | 中国农业科学院植物保护研究所 | Taqman-MGB detection method of the Plasmopara viticola to carboxylic acyloxy amine fungicide resistance |
| CN111893204A (en) * | 2020-08-14 | 2020-11-06 | 岭南师范学院 | Primer, kit and method for detecting high-resistance benzimidazole bactericide genotype E198A lawn scab |
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| CN103820563A (en) * | 2014-03-12 | 2014-05-28 | 南京农业大学 | LAMP (loop-mediated isothermal amplification)-based method for rapid detection of sclerotinia sclerotiorum strain with high resistance to carbendazim |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103820563A (en) * | 2014-03-12 | 2014-05-28 | 南京农业大学 | LAMP (loop-mediated isothermal amplification)-based method for rapid detection of sclerotinia sclerotiorum strain with high resistance to carbendazim |
Non-Patent Citations (2)
| Title |
|---|
| YA-BING DUAN等: "development and evaluation of a novel and rapid detection assay for botrytis cinerea based on loop-mediated isothermal amplification", 《PLOS ONE》, 20 October 2014 (2014-10-20) * |
| 刘圣明: "灰葡萄孢菌抗多菌灵β-微管蛋白基因在禾谷镰孢菌种的表达研究", 《中国博士学位论文全文数据库农业科技辑》, 15 July 2012 (2012-07-15), pages 046 - 13 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104911258A (en) * | 2015-04-23 | 2015-09-16 | 南京农业大学 | Carbendazim resistant genotype F200Y sclerotinia sclerotiorum LAMP detection primer composition and LAMP test kit and LAMP test method |
| CN108315472A (en) * | 2018-04-27 | 2018-07-24 | 安徽省农业科学院植物保护与农产品质量安全研究所 | It is a kind of to be used for the Primer composition and its application that alternaric bacteria LAMP is quickly detected |
| CN109666754A (en) * | 2018-11-26 | 2019-04-23 | 中国农业科学院植物保护研究所 | Taqman-MGB detection method of the Plasmopara viticola to carboxylic acyloxy amine fungicide resistance |
| CN111893204A (en) * | 2020-08-14 | 2020-11-06 | 岭南师范学院 | Primer, kit and method for detecting high-resistance benzimidazole bactericide genotype E198A lawn scab |
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